4-3 The mechanics of dilution plating

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Typical bacterial populations in a 1 ml or 1 g sample can be as high as 1011 CFU's. If most samples were spread directly onto a plate, there would be so many organisms that individual colonies would not form and a viable count would be impossible. Therefore, samples almost always need to be diluted. Using simple 1/10 or 1/100 dilutions is most convenient and makes later calculations easier. For a 1/10 dilution 1 part sample is added to 9 parts diluent. Note this is not 1 part to 10 parts (that would be a 1/11 dilution)! For a 1/100 dilution, 1 part is added to 99 parts.

When a known amount of a sample is added by pipette to a known amount of diluent (called a dilution blank) the resulting suspension must be thoroughly mixed. Rolling a tube between the hands is not as effective as giving the tube a series of firm flicks with the index finger. (End-to-end mixing is not done with the cotton plugged or capped tubes because they will leak.) A series of dilutions may be made as desired, and from one or more of these dilutions, known amounts are inoculated onto the surface of agar media in plates. Care must be taken to open the plates just enough to admit the pipette, in order to decrease the risk of contamination.

Following the placement of the inoculum on the surface of the plate, a sterile, L-shaped glass rod (often called a "hockey stick") is used to spread the inoculum over the entire surface of the medium. The part of the rod which enters the plate is sterilized by immersion in 95% ethanol followed by passing the rod through a flame such that the ethanol can burn off. (It should be noted that one such flaming may not always kill all of the endospores which may be present on the rod!) After a short period of cooling, the rod is ready for use.

Once diluents are prepared and then added to a plate, the medium is incubated at an appropriate temperture to allow growth of the microorganisms. Colonies arising on the plate can then be enumerated, and this count used to calculate the total number microbe present in the original sample.

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